Kindlin-2 suppresses transcription factor GATA4 through interaction with SUV39H1 to attenuate hypertrophy

Figures

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  Fig. 1. Kindlin-2 suppresses expression of GATA4. a GO analysis of differentially expressed genes in RNAseq. b 11 differentially expressed genes (log2 Fold > 1) in cardiovascular disease-related genes. c Control or Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes for 48 h, followed western blot. d Protein bands were scanned and relative band intensities were normalized to each GAPDH band. The column diagrams represent average relative band intensity with standard error from three independent experiments. e Cardiomyocytes were infected by Kindlin-2 or control adenovirus, followed western blot. f Relative band intensities were analyzed. g Control or Kindlin-2 siRNA was transfected into cardiomyocytes and RT-qPCR detected Kindlin-2 and GATA4 mRNA level. h Adenovirus carrying Kindlin-2 or control was transfected into cardiomyocytes, followed RT-qPCR. i Schematic diagrams of the regions for ChIP assay. j Lysates were extracted from mouse cardiac tissues for ChIP assays using anti-Kindlin-2 antibody. Q-PCR assay was then performed to quantify ChIP-enriched DNA using the three primers.

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  Fig. 2. SUV39H1 mediated suppression of Kindlin-2 on GATA4. a Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by Western blot. b Relative band intensities of western blot were analyzed. c Flag or Flag-SUV39H1 was transfected into HEK293A cells and GATA4 were measured by RT-qPCR. d HEK293A cells were treated with the SUV39H1 inhibitor Chaetomin at various doses for 48 h, and GATA4 was measured by western blot. e Relative band intensities of western blot were analyzed. f HEK293A cells were treated with Chaetomin and GATA4 was detected by RT-PCR. g Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. Means ± S.D. ** indicates p < 0.01, *** indicates p < 0.001.

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  Fig. 3. Kindlin-2 interacts with SUV39H1. a HEK293A cells were treated by Chaetomin and lysates were then extracted for ChIP assays. b Flag-SUV39H1 was transfected into HEK293A cells and lysates were then extracted for ChIP assays. c Lysates from HEK293A cells were prepared and then anti-Kindlin-2 antibody was used for Co-IP. d Purified GST or GST-SUV39H1 was incubated with total lysates prepared from HEK293A cells for the GST-pull down assays. e Immunofluorescent staining of anti-Kindlin-2 (Alexa Fluor 488, green), anti-SUV39H1 (Alexa Fluor 568, red), and nuclei (DAPI, blue) was performed using primary neonate rat cardiomyocytes. Images were captured with a confocal microscope (Scale bar = 20 µm). Pearson’s coefficient was shown in the histogram.

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  Fig. 4. Kindlin-2 prevents cardiomyocytes from hypertrophy through suppressing GATA4. a–c Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO treatment (5 µm, 24 h). The efficacy of Kindlin-2 overexpression was detected by Western blot a. Representative images of cardiomyocytes stained with anti-α-actinin2 (Alexa Fluor 568) b. Cell surface areas were measured c. d–f Kindlin-2 siRNA was transfected into primary neonate rat cardiomyocytes plus ISO treatment with or without knockdown of GATA4 and GATA6 alone or in combination. The efficacy of Kindlin-2 siRNA was detected by Western blotting d. Representative images of cardiomyocytes stained with anti-α-actinin2 e. Cell surface areas were measured f. g Cardiomyocytes were infected with Kindlin-2 or control adenovirus plus ISO, followed by Western blot (left panel). Relative band intensities were analyzed (right panel). h Cardiomyocytes were transfected with control or Kindlin-2 siRNA plus ISO treatment, followed Western blot (left panel). Relative band intensities were analyzed (right panel).

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  Fig. 5. Cardiac-specific Kindlin-2 knockout leads to hypertrophy. a Strategy for generating myocardial conditional knockout Kindlin-2 mice. The targeting construct was designed to insert loxP sites flanking exons 5 and 6 of Kindlin-2 gene. Kindlin-2 floxed mice were then crossed with mice expressing recombinant α- MHC-Cre mice to produce Kindlin-2 myocardial conditional knockout (cKO) mice. b PCR analysis of extracted genomic DNA from tail clippings. c–e Western blot, RT-qPCR and immunohistochemical staining showed the level of Kindlin-2 in heart of wild-type or cKO mice (Scale bar = 50 µm). f Representative macroscopic observations of hearts (upper panel). HE staining of whole-heart sections (lower panel) (Scale bar = 1000 µm). g Ratio of heart weight to body weight or heart weight to tibia length was determined in saline or ISO-treated mice. Means ± S.D. *p < 0.05. h HE staining of left ventricular muscle sections (Scale bar = 50 µm) (left panel). The surface areas of cardiomyocyte were quantified (right panel). Means ± S.D. *p < 0.05. i Staining of left ventricular muscle sections with cell membrane probe wheat germ agglutinin (Scale bar = 50 µm) (left panel).The average diameter of…

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  Fig. 6. Activation of GATA4 mediates cardiac hypertrophy in Kindlin-2 cKO mice. a Wild-type Kindlin-2 or cKO mice were injected with saline or ISO. Total protein was extracted from heart tissue for western blot (left panel). Protein bands on the left were scanned and relative band intensities were normalized (right panel). b Immunohistochemical staining showed GATA4 level (Scale bar = 50 µm). c Lysates from heart tissue of wild-type mice were prepared for Co-IP. d Lysates were extracted from heart tissue of wild-type and Kindlin-2 cKO mice for ChIP assays. Means ± S.D. *P < 0.05, **p < 0.01.

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  Fig. 7. A working model for the role of Kindlin-2 in suppressing GATA4.